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Gene expression of cell diferenciation related factors of respiratory epithelium and its roll in the mucosal pathology of rhino sinusitis

鼻窦炎黏膜病理中呼吸道上皮及其卷曲细胞分化相关因子基因表达

基本信息

批准号：
09671755
负责人：
MATSUNE Shoji
金额：
$ 1.41万
依托单位：
Kagoshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Genetic investigation : Several bands that appeared on the collagen gel following floating were extracted by the differential display method. The protein volume expression of 5 of the 25 sequenced bands was determined before and after floating by Northern blotting. Protein expression did not differ between the two measurement period. This lack of difference was attributed to the high frequency of false positive results using Northern blotting.Morphological confirmation of cell differentiation : Expression of cytokeratin the cultured cells was examined chronologically by an immunohistochemical technique using anti-cytokeratin 8, 13 and 14 monochronal antibodies, although the cultured cells showed no positive findings before floating, this analysis showed a gradual increase in positive staining after floating in the following pattern ; anti-cytokeratin 8 was positively stained from the middle to apical layer of the cultured cells and anti-cytokeratin 13 was positively stained only in the middle layer and anti-cytokeratin 14 was positively stained in the basal layer.Detection of ciliogenesis-specific substance : Several monochronal antibodies were produced, of which RPCS3a, the most eligible candidate specific to ciliogenesis, was characterized. RPCS3a was expressed in the cultured cell from the 3rd day after floating according to cellular ELISA.On the 10th day, RPCS3a was expressed most strongly, and many cultured cells contained secretory vesicles at their apical site as well as numerous centrioles indicating to be preciliated cells. ELISA revealed that RPCS3a is specific to the a sort of fucosylated carbohydrate H type 3. The expressed volume of H type 3 recognized by RPCS3a appears to reflect the stage of ciliogenesis. Based on the present data, we consider H type 3 to be a relativity specific markers of ciliogenesis and thus may provide a useful tool to clarify the mechanisms of ciliogenesis.

遗传学研究：用差异显示法提取漂浮后胶原凝胶上出现的条带。用Northern blotting法测定25个测序条带中5个条带漂浮前后的蛋白体积表达量。蛋白表达在两个测量期间无差异。这种差异的缺乏是由于使用Northern印迹法产生假阳性结果的频率很高。细胞分化的形态学证实：使用抗细胞角蛋白8、13和14单时抗体，用免疫组织化学技术按时间顺序检测培养细胞的细胞角蛋白表达，尽管培养细胞在漂浮前未见阳性结果，但该分析显示，漂浮后阳性染色逐渐增加，呈以下模式：抗细胞角蛋白8在培养细胞的中层至顶层呈阳性染色，抗细胞角蛋白13仅在中层呈阳性染色，抗细胞角蛋白14在基底层呈阳性染色。纤毛发生特异性物质的检测：产生了几种单克隆抗体，其中RPCS3a是最适合纤毛发生特异性的候选抗体。根据细胞ELISA法，RPCS3a在浮后第3天开始在培养细胞中表达。在第10天，RPCS3a表达最强烈，许多培养细胞的顶端含有分泌囊泡和大量的中心粒，表明它们是早熟细胞。ELISA结果显示，RPCS3a对一种聚焦型碳水化合物h3型具有特异性。RPCS3a识别的H型3的表达量似乎反映了纤毛发生的阶段。基于目前的数据，我们认为h3型是一种相对特异性的纤毛发生标志物，因此可能为阐明纤毛发生机制提供有用的工具。